Method of treating hypertrophic cardiomyopathy

ABSTRACT

We found that FIZZ1/RELMα is inducible by hypoxia in lung. The hypoxia-upregulated expression of FIZZ1/RELMα was located in the pulmonary vasculature, bronchial epithelial cells, and type II pneumocytes. Recombinant FIZZ1/RELMα protein stimulates rat pulmonary microvascular smooth muscle cell (RPSM) proliferation dose-dependently. Therefore, we renamed this gene as hypoxia-induced mitogenic factor (HIMF). HIMF strongly activated Akt phosphorylation. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 inhibits HIMF-activated Akt phosphorylation. It also inhibits HIMF-stimulated RPSM proliferation. Thus, the PI3K/Akt pathway, at least in part, mediates the proliferative effect of HIMF. HIMF also has angiogenic and vasoconstrictive activity. Notably, HIMF increases pulmonary arterial pressure and vascular resistance more potently than either endothelin-1 or angiotensin II.

This application was made using funds from National Institutes of Healthgrant HL 39706. The U.S. government therefore retains certain rights inthe invention.

FIELD OF THE INVENTION

The invention relates to vasoconstriction, neoangiogenesis, mitogenesis,and vascular remodeling. In particular it relates to an endogenousbiological factor that affects these important disease-relatedprocesses.

BACKGROUND OF THE INVENTION

FIZZ1 (found in inflammatory zone 1), is a protein induced in murinelung in an ovalbumin-induced asthma model.¹ Besides its induction in thebronchial mucosal epithelial cells, FIZZ1 was also induced in type IIpneumocytes and it inhibited NGF-induced survival of DRG neurons andNGF-mediated increase in neuronal CGRP content.¹ Holcomb et al alsoreported that FIZZ1 is a secreted protein sharing the consensus sequenceof 10 cysteine residues in the C-terminus (¹CX₁₁ ²CX₈ ³CX⁴CX₃ ⁵CX₁₀⁶CX⁷CX⁸CX₉ ⁹C¹⁰C) with two other murine genes expressed respectively inintestinal crypt epithelial (FIZZ2) and white adipose tissue (FIZZ3) andtwo related human genes (human FIZZ1 and human FIZZ3).¹ Later, FIZZ3 wasshown to be implicated in type II diabetes mellitus and was renamed asresistin.² FIZZ1 and FIZZ2 were renamed as resistin-like molecule α(RELMα) and β (RELMβ), respectively.³ Human FIZZ1, however, was renamedas human RELMβ.³ Recently, FIZZ1 was found in macrophages⁴ and in thestromal vascular fraction of adipose tissue,⁵ and it inhibited adipocytedifferentiation.⁶ However, the function of FIZZ1 remained unclear.

There is a need in the art for effective methods of treating pulmonaryhypertension, cancer, cardiac hypertrophy, cardiac ischemia, wounds, andother diseases relating to neoangiogenesis and vasoconstriction.

BRIEF SUMMARY OF THE INVENTION

According to one embodiment of the invention method is provided ofpromoting wound healing in a patient with a wound. A wound-healingamount of a HIMF protein comprising the amino acid sequence shown in SEQID NO: 1 is administered to a patient in need thereof.

According to another embodiment of the invention a method is provided ofpromoting wound healing in a patient with a wound. A wound-healingamount of an expression construct that encodes HIMF (SEQ ID NO: 1) isadministered to a patient in need thereof.

According to yet another embodiment of the invention a method isprovided of screening for therapeutic agents. A test agent is contactedwith HIMF-treated pulmonary smooth muscle cells. Phosphorylation of Aktis determined A test agent which inhibits HIMF-activated phosphorylationof Akt in the pulmonary smooth muscle cells is identified as a candidatetherapeutic agent.

According to an additional embodiment of the invention a method isprovided of treating diabetic retinopathy. An antibody whichspecifically binds to a protein comprising the sequence of SEQ ID NO: 1is administered to a patient in need thereof in an amount sufficient torelieve symptoms of diabetic retinopathy.

According to yet another embodiment of the invention a method isprovided of treating a tumor. An antibody which specifically binds to aprotein comprising the sequence of SEQ ID NO: 1 is administered to apatient in need thereof, in an amount sufficient to stop tumorprogression.

According to still another embodiment of the invention a method isprovided of treating diabetic retinopathy LY294002 is administered to apatient in need thereof, in an amount sufficient to relieve diabeticretinopathy.

According to one more embodiment of the invention a method is providedof treating tumors. LY294002 is administered to a patient in needthereof, in an amount sufficient to stop tumor progression.

According to even another embodiment of the invention a method isprovided of treating asthma. LY294002 is administered to a patient inneed thereof, in an amount sufficient to relieve asthma-associatedsymptoms.

According to an embodiment of the invention a method is provided oftreating pulmonary hypertension. LY294002 is administered to a patientin need thereof, in an amount sufficient to relieve pulmonaryhypertension.

According to yet another embodiment of the invention a method isprovided of treating a patient who has or is at risk of heart ischemia.An antibody which specifically binds to a protein comprising thesequence of SEQ ID NO: 1 is administered to the patient in an amountsufficient to reduce the extent of tissue damage to the heart.

According to another embodiment of the invention a method is provided oftreating a patient who has or who is at risk of heart ischemia. LY294002is administered to the patient in an amount sufficient to reduce theextent of tissue damage to the heart.

According to one more embodiment of the invention a method is providedof treating a patient with hypertrophic cardiomyopathy. An antibodywhich specifically binds to a protein comprising the sequence of SEQ IDNO: 1 is administered to the patient in an amount sufficient to reducethe extent of hypertrophic cardiomyopathy.

According to one embodiment of the invention a method is provided oftreating a patient with hypertrophic cardiomyopathy. LY294002 isadministered to the patient in an amount sufficient to reduce the extentof hypertrophic cardiomyopathy.

According to yet another embodiment of the invention a method oftreating diabetic retinopathy is provided. An antisense constructcomprising at least 15 nucleotides of a human HIMF cDNA is delivered tothe patient, whereby cells of the patient's retina express an mRNAmolecule which is complementary to native HIMF mRNA.

According to one embodiment of the invention a method is provided oftreating diabetic retinopathy.

An RNA interference construct comprising at least 19 nucleotides of ahuman HIMF cDNA is administered to a patient with diabetic retinopathy,whereby cells of the patient's retina express a double stranded RNAmolecule, one of whose strands is complementary to native HIMF mRNA.

According to one additional embodiment of the invention a method oftreating diabetic retinopathy is provided. siRNA comprising 19 to 21 bpduplexes of a human HIMF mRNA with 2 nt 3′ overhangs is delivered to apatient with diabetic retinopathy, whereby HIMF mRNA produced by thepatient's retina cells is cleaved.

According to one embodiment of the invention a method is provided fortreating diabetic retinopathy. An antisense oligonucleotide comprisingat least 15 nucleotides of a human HIMF cDNA is delivered to a patientwith diabetic retinopathy, whereby cells of the patient's retina expressan mRNA molecule which is complementary to native HIMF mRNA.

According to one embodiment of the invention a method of treating atumor in a patient is provided. An antisense construct comprising atleast 15 nucleotides of a human HIMF cDNA is delivered to a tumor cell,whereby the tumor cell expresses an mRNA molecule which is complementaryto native HIMF mRNA.

According to one embodiment of the invention a method is provided oftreating a tumor in a patient. An RNA interference construct comprisingat least 19 nucleotides of a human HIMF cDNA is administered to a tumorcell in a patient, whereby the tumor cell expresses a double strandedRNA molecule one of whose strands is complementary to native HIMF mRNA.

According to another embodiment of the invention a method is provided oftreating tumor in a patient. An siRNA comprising 19 to 21 bp duplexes ofa human HIMF mRNA with 2 nt 3′ overhangs is administered to a tumor cellin a patient, whereby HIMF mRNA produced by the tumor cell is cleaved.

According to one more embodiment of the invention a method is providedof treating a patient with a tumor. An antisense oligonucleotidecomprising at least 15 nucleotides of a human HIMF cDNA is administeredto a tumor cell in a patient, whereby the tumor cell expresses an mRNAmolecule which is complementary to native HIMF mRNA.

According to one additional embodiment of the invention a method isprovided of treating pulmonary hypertension. An antisense constructcomprising at least 15 nucleotides of a human HIMF cDNA is delivered tolung cells of a patient with pulmonary hypertension, whereby thepatient's lung cells express an mRNA molecule which is complementary tonative HIMF mRNA.

According to a further embodiment of the invention a method of treatingpulmonary hypertension is provided. An RNA interference constructcomprising at least 19 nucleotides of a human HIMF cDNA is delivered toa lung cell of a patient with pulmonary hypertension, whereby the lungcell expresses a double stranded RNA molecule one of whose strands iscomplementary to native HIMF mRNA.

According to still another embodiment of the invention a method oftreating pulmonary hypertension is provided. siRNA comprising 19 to 21bp duplexes of a human HIMF mRNA with 2 nt 3′ overhangs is delivered toa lung cell of a patient with pulmonary hypertension, whereby HIMF mRNAproduced by the lung cell is cleaved.

According to one additional embodiment of the invention a method oftreating pulmonary hypertension is provided. An antisenseoligonucleotide comprising at least 15 nucleotides of a human HIMF cDNAis delivered to a lung cell of a patient with pulmonary hypertension,whereby the lung cell expresses an mRNA molecule which is complementaryto native HIMF mRNA.

According to even another embodiment of the invention a method oftreating a patient who has or who is at risk of heart ischemia isprovided. An antisense construct comprising at least 15 nucleotides of ahuman HIMF cDNA is delivered to a heart cell of the patient, whereby theheart cell expresses an mRNA molecule which is complementary to nativeHIMF mRNA.

According to one aspect of the invention a method of treating a patientwho has or who is at risk of heart ischemia is provided. An RNAinterference construct comprising at least 19 nucleotides of a humanHIMF cDNA is delivered to a heart cell of the patient, whereby the heartcell expresses a double stranded RNA molecule one of whose strands iscomplementary to native HIMF mRNA.

According to one aspect of the invention a method of treating a patientwho has or who is at risk of heart ischemia is provided. siRNAcomprising 19 to 21 bp duplexes of a human HIMF mRNA with 2 nt 3′overhangs is delivered to a heart cell of the patient, whereby HIMF mRNAproduced by the heart cell is cleaved.

According to another aspect of the invention a method of treating apatient who has or who is at risk of heart ischemia is provided. Anantisense oligonucleotide comprising at least 15 nucleotides of a humanHIMF cDNA is delivered to a heart cell of the patient, whereby the heartcell expresses an mRNA molecule which is complementary to native HIMFmRNA.

According to another aspect of the invention a method of treatinghypertrophic cardiomyopathy is provided. An antisense constructcomprising at least 15 nucleotides of a human HIMF cDNA is delivered toa heart cell of a patient with hypertrophic cardiomyopathy, whereby theheart cell expresses an mRNA molecule which is complementary to nativeHIMF mRNA.

According to still one more aspect of the invention a method of treatinghypertrophic cardiomyopathy is administered. An RNA interferenceconstruct comprising at least 19 nucleotides of a human HIMF cDNA isdelivered to a heart cell of a patient with hypertrophic cardiomyopathy,whereby the heart cell expresses a double stranded RNA molecule one ofwhose strands is complementary to native HIMF mRNA.

According to even one more aspect of the invention a method of treatinghypertrophic cardiomyopathy is provided. siRNA comprising 19 to 21 bpduplexes of a human HIMF mRNA with 2 nt 3′ overhangs is delivered to aheart cell of a patient with hypertrophic cardiomyopathy, whereby HIMFmRNA produced by the heart cell is cleaved.

According to still one more aspect of the invention a method of treatinghypertrophic cardiomyopathy is provided. An antisense oligonucleotidecomprising at least 15 nucleotides of a human HIMF cDNA is delivered toa heart cell of a patient with hypertrophic cardiomyopathy, whereby theheart cell expresses an mRNA molecule which is complementary to nativeHIMF mRNA.

A method is provided for promoting proliferation and differentiation ofstem cells. An effective amount of a HIMF protein comprising the aminoacid sequence shown in SEQ ID NO: 1 is administered to a population ofstem cells. The stem cells thereby proliferate and/or differentiate.

A method is provided for promoting proliferation and differentiation ofstem cells. An effective amount of an expression construct that encodesHIMF (SEQ ID NO: 1) is administered to a population of stem cells. Thestem cells thereby proliferate and/or differentiate.

A method is provided for promoting proliferation of vascular endothelialcells. An effective amount of a HIMF protein comprising the amino acidsequence shown in SEQ ID NO: 1 is administered to a population ofvascular endothelial cells. The vascular endothelial cells therebyproliferate.

A method is provided for promoting proliferation of vascular endothelialcells. An effective amount of an expression construct that encodes HIMF(SEQ ID NO: 1) is administered to a population of vascular endothelialcells. The vascular endothelial cells thereby proliferate.

These and other embodiments of the invention provide the art withmethods of alleviating symptoms and causes of serious diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1D show hypoxia induction of HIMF. FIG. 1A shows a timecourse of HIMF mRNA expression in mouse lung. N, H1d, H4d, H7d, and H3wrepresent mouse lung exposure to room air (normoxia) or to 10% O₂(hypoxia) for 1, 4, 7, and 21 days, respectively. *P<0.05 vs N. FIG. 1Bshows a western blot of HIMF. Samples are from mice exposed to normoxiaor hypoxia for 4 days. Immunohistochemical staining of HIMF protein inmouse lungs exposed to normoxia (FIG. 1C) or to hypoxia for 4 days (FIG.1D).

FIG. 2A shows dose-response curve of HIMF-stimulated ³H-thymidineincorporation in passage-3 RPSM (5 separate experiments, n=9 to 18).Inset, RPSM were untreated (none) or treated with HIMF (5×10⁻⁸ mol/L) orwith FLAG. *P<0.001 vs untreated. FIG. 2B shows passage-4 RPSM treatedwith HIMF (5×10⁻⁸ mol/L) or with FLAG. The lysates were subject toWestern blot for pAkt and Akt. FIG. 2C shows passage-4 RPSM treated withFLAG or HIMF (5×10⁻⁸ mol/L) in the absence or presence of genistein (20μmol/L), NF449 (50 μmol/L), or LY294002 (10 μmol/L) for 5 minutes. Thelysates were subjected to Western blot for pAkt and Akt. FIG. 2D showspassage-4 RPSM treated with HIMF (5×10⁻⁸ mol/L) or FLAG in the absence(Control) or presence of LY294002 (10 μmol/l or 30 μmol/L). n=9 from 4separate experiments; P<0.001 vs Control.

FIGS. 3A-3E shows stimulation of vascular tube formation. Matrigel plugscontaining FLAG were stained either with hematoxylin and eosin (HE)(FIG. 3A) or von Willebrand factor (vWF) (FIG. 3B), and matrigel plugscontaining HIMF (5×10⁻⁸ mol/L) were stained with either HE (FIG. 3C) orvWF (FIG. 3D). The arrowheads point to the newly formed vascular tubesin the matrigel plug. FIG. 3E shows dose-response curves of HIMF andvasoconstricting agents U-46619, endothelin-1 (ET-1), angiotensin II(Ang II), and serotonin (5-HT). The insets are the volume-response curveof the FLAG elution buffer and the graphs of HIMF perfusion on PAP andPVR.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present inventors that HIMF is ahypoxia-induced mitogenic factor in lung with potent angiogenic andpulmonary vasoconstrictive properties. The PI3K/Akt pathway, at least inpart, mediates its proliferative (mitogenic) effect. These propertiesrender HIMF a useful tool for stimulating wound healing and makeinhibition of HIMF a useful tool for treating such disease conditions asdiabetic retinopathy, tumors, pulmonary hypertension (with or withoutallergic or inflammatory involvement), heart ischemia, and hypertrophiccardiomyopathy. Inhibition of HIMF can be done by any method known inthe art. These include, without limitation, use of antibodies, antisenseoligonucleotides, antisense constructs, RNA interference constructs orsiRNA duplex RNA molecules, each of which is specific for HIMF proteinor mRNA. Inhibitors of downstream events in the HIMF pathway can also beused, as described below.

As is known in the art, supplying a protein to a cell can be donedirectly by delivering the protein or can be done indirectly bydelivering a polynucleotide that encodes the protein. Either method canbe used to treat wounds. The protein and/or polynucleotide can bedelivered by any means known in the art. It can, for example, bedelivered locally to the wounded area. It can alternatively be deliveredintravenously. Other methods of delivery, such as oral, peritoneal,subcutaneous, intramuscular, intradermal, and topical, can be used aswell. Proteins and/or polynucleotides can be formulated by any meansknown in the art. For example, liposomes can be used to deliver eitherproteins or polynucleotides. The coding sequence for HIMF protein can becontained in any useful vector known in the art. Viral or plasmidvectors can be used, for example. Wounds which are amenable to HIMFtreatment include, without limitation, those that are caused by injuryor surgery. The HIMF that is delivered may be the mature form of theprotein or the precursor form. A mature form of the protein in shown inSEQ ID NO: 1 and a precursor form is shown in SEQ ID NO: 2. Proteolyticcleavage converts the precursor form to the mature form. A fusion orconjugated protein comprising the mature protein or precursor proteinwith additional moieties attached can also be used. For example,moieties with similar or complementary activities can be fused orconjugated to the HIMF sequence.

HIMF protein or polynucleotides encoding its coding sequence can also beadministered in vitro or in vivo to stem cells to promote theirproliferation and/or differentiation. For example, HIMF accelerates therate of differentiation of isolated stem cells into striated muscle. Inaddition, HIMF protein or polynucleotides encoding its coding sequencecan be used to stimulate proliferation of vascular endothelial cells.This can be of use to generate sufficient cells of appropriate type touse for tissue engineering purposes, in particular in creating bloodvessels. The blood vessels can be used for example, for coronary bypassor replacement of stenosed vessels.

LY294002 is one inhibitor of HIMF-mediated events which is useful fortreating diabetic retinopathy, tumors, pulmonary hypertension, heartischemia, and hypertrophic cardiomyopathy. LY294002, [CAS number154447-36-6] also known as2-(4-morpholinyl)-8-phyenyl-4H-1-benzopyran-4-one or2-(4-morpholinyl)-8-phyenylchromone, is a specific inhibitor ofphosphatidylinosotol 3-kinase. Vlahos et al., J. Biol. Chem. 1994,269:5241-5248. LY294002 inhibits the phosphorylation of Akt.

Antisense constructs, antisense oligonucleotides, RNA interferenceconstructs or siRNA duplex RNA molecules can be used to interfere withexpression of HIMF. Typically at least 15, 17, 19, or 21 nucleotides ofthe complement of HIMF mRNA sequence are sufficient for an antisensemolecule. Typically at least 19, 21, 22, or 23 nucleotides of HIMF aresufficient for an RNA interference molecule. Preferably an RNAinterference molecule will have a 2 nucleotide 3′ overhang. If the RNAinterference molecule is expressed in a cell from a construct, forexample from a hairpin molecule or from an inverted repeat of thedesired HIMF sequence, then the endogenous cellular machinery willcreate the overhangs. siRNA molecules can be prepared by chemicalsynthesis, in vitro transcription, or digestion of long dsRNA by RnaseIII or Dicer. These can be introduced into cells by transfection,electroporation, or other methods known in the art. See Hannon, G J,2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002,The rest is silence. RNA 7: 1509-1521; Hutvagner G et al., RNAi: Natureabhors a double-strand. Curr. Opin. Genetics & Development 12: 225-232;Brummelkamp, 2002, A system for stable expression of short interferingRNAs in mammalian cells. Science 296: 550-553; Lee N S, Dohjima T, BauerG, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J. (2002). Expressionof small interfering RNAs targeted against HIV-1 rev transcripts inhuman cells. Nature Biotechnol. 20:500-505; Miyagishi M, and Taira K.(2002). U6-promoter-driven siRNAs with four uridine 3′ overhangsefficiently suppress targeted gene expression in mammalian cells. NatureBiotechnol. 20:497-500; Paddison P J, Caudy A A, Bernstein E, Hannon GJ, and Conklin D S. (2002). Short hairpin RNAs (shRNAs) inducesequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958;Paul C P, Good P D, Winer I, and Engelke D R. (2002). Effectiveexpression of small interfering RNA in human cells. Nature Biotechnol.20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester W C, andShi Y. (2002). A DNA vector-based RNAi technology to suppress geneexpression in mammalian cells. Proc. Natl. Acad. Sci. USA99(6):5515-5520; Yu J-Y, DeRuiter S L, and Turner D L. (2002). RNAinterference by expression of short-interfering RNAs and hairpin RNAs inmammalian cells. Proc. Natl. Acad. Sci. USA 99(9):6047-6052.

Antisense or RNA interference molecules can be delivered in vitro tocells or in vivo, e.g., to tumors, eyes, lungs, bronchia, heart, orretina of a mammal. Typical delivery means known in the art can be used.For example, delivery to a tumor can be accomplished by intratumoralinjections. Delivery to lung can be accomplished by instillation. Othermodes of delivery can be used without limitation, including:intravenous, intramuscular, intraperitoneal, intraarterial, localdelivery during surgery, endoscopic, subcutaneous, and per os. In amouse model, the antisense or RNA interference can be adminstered to atumor cell in vitro, and the tumor cell can be subsequently administeredto a mouse. Vectors can be selected for desirable properties for anyparticular application. Vectors can be viral or plasmid. Adenoviralvectors are useful in this regard. Tissue-specific, cell-type specific,or otherwise regulatable promoters can be used to control thetranscription of the inhibitory polynucleotide molecules. Non-viralcarriers such as liposomes or nanospheres can also be used.

HIMF stimulates pulmonary smooth muscle cells and cardiac myocytes tophosphorylate Akt. This is mediated by a phosphatidylinositol 3-kinase(PI3K) enzyme. Inhibitors of this phosphorylation can be usedtherapeutically to treat diseases such as diabetic retinopathy, tumors,pulmonary hypertension, heart ischemia, and hypertrophic cardiomyopathy.Inhibitors can be found by screening test agents by contacting them withHIMF-treated pulmonary smooth muscle cells. Alternatively, the testagent may be contacted with a cell-free, in vitro system which comprisesphosphatidylinositol 3-kinase (PI3K) and Akt. The extent ofphosphorylation of Akt can be determined in the presence and absence ofthe test agent. A test agent which inhibits HIMF-activatedphosphorylation of Akt in the pulmonary smooth muscle cells or in thecell-free system can be identified as a candidate therapeutic agent.Phosphorylation of Akt can be determined by any means known in the art.One convenient means employs western blot. Further testing in othermodel systems or in clinical settings may be performed to confirminhibitory activity and/or extent of toxicity

While the invention has been described with respect to specific examplesincluding presently preferred modes of carrying out the invention, thoseskilled in the art will appreciate that there are numerous variationsand permutations of the above described systems and techniques that fallwithin the spirit and scope of the invention as set forth in theappended claims.

EXAMPLES Example 1 Materials and Methods

Animals. Sprague-Dawley rats were purchased from Hilltop Lab Animal Inc.(Scottdale, Pa.), and C57/BL6 mice were obtained from Charles RiverLaboratories (Wilmington, Mass.). Housing and procedures involvingexperimental animals were approved by the Animal Care and Use Committeeof the Johns Hopkins University.

Hypoxia exposure. The protocol for the exposure of animals to hypoxiahas been previously described1. Briefly, C57BL6 mice aged 8-12 weekswere placed in a Plexiglas chamber maintained at 10% O2 (hypoxic group)or in a chamber open to room air (normoxic group) for specific timeswith a 12:12-h light-dark cycle. Hypoxia was maintained using a Pro:oxmodel 350 unit (Reming Bioinstruments, Refield, N.Y.), which controlledfractional concentration of O2 in inspired gas by solenoid-controlledinfusion of N2 (Roberts Oxygen, Rockville, Md.) balanced against aninward leak of air through holes in the chamber. The hypoxic mice wereexposed to room air for about 10 min daily while their cages werechanged. CO2, water vapor, and ammonia were removed by pumping theatmosphere of the hypoxia chamber through Bara Lyme (barium hydroxidelime, USP; Chemetron Medical Division, Allied Healthcare Products, St.Louis, Mo.), Drierite (anhydrous calcium sulfate; Fisher Scientific,Atlanta, Ga.), and activated carbon (Fisher Scientific).

Cell culture and ³H-thymidine incorporation assays. Rat pulmonarymicrovascular smooth muscle cells (RPSM) were isolated from maleSprague-Dawley rats (150-200 g) according to a published method2.Briefly, after the pulmonary artery was flushed with phosphate bufferedsaline (PBS), it was infused with 2% iron oxide suspension in 0.5% agar.The air spaces were filled with agar, the subpleural margins wereharvested and partially collagenase digested, and the microvessels wereseparated magnetically. The identity of RPSM was confirmed bycharacteristic appearance on phase-contrast microscopy and indirectimmunofluorescent antibody staining for smooth muscle-specific isoformsof β-actin and myosin heavy chain. RPSM were grown in DMEM/F12 mediumsupplemented with 10% fetal bovine serum (FBS), 2 mM of glutamine, 100U/ml of penicillin and 100 μg/ml of streptomycin. Passage 3 to 5 cellswere used in the experiments.

The cells were seeded in 24-well plates at 5×104/well. Aftersynchronization with serum-free DMEM/F12 (supplemented with 1% Bovineserum albumin (BSA)) for 24 to 48 hours, the cells were treated with thespecified agents and 0.1 μCi/well (or 1 μCi/well) of 3H-thymidine in theserum-free DMEM/F12 for 48 hours. After the experimental protocol wascompleted, the cells were rinsed with PBS twice and incubated in 10% TCAfor 30 min. The cells were then washed with PBS two more times and lysedin 250 μl of the lysis buffer (1% SDS, 0.8% NaOH). 3H-thymidineincorporation was measured with a Beckman 6500 scintillation counter in10 ml of scintillation cocktail.

RT-PCR. One step RT-PCR kit from Qiagen (Valencia, Calif.) was used forthe duplex RT-PCR. 0.8 μM each of primers 5′-CAA TCC CAT GGC GTA TAA AAGCAT C-3′ (HIMF forward; SEQ ID NO: 5) and 5′-TCA TTC TTA GGA CAG TTG GCAGCA G-3′ (HIMF reverse; SEQ ID NO: 6), 0.05 μM each of primers 5′-AAGATG ACC CAG ATC ATG TTT GAG ACC-3′ (β-actin forward; SEQ ID NO: 7) and5′-GAG CAA TGA TCT TGA TCT TCA TGG TG-3′ (β-actin reverse; SEQ ID NO:8), and 1 μg of total RNA isolated from the murine lung with Tri Reagent(Sigma, St. Louis, Mo.) were used in 50 μl reactions in an iCycler(BioRad, Hercules, Calif.) with the following cycling parameters: 50° C.for 30 min, 95° C. for 15 min, and 28 cycles of 94° C. for 1 min, 55° C.for 30 seconds, and 72° C. for 1 min, followed by 72° C. for 7 min.

Recombinant HIMF. The C-terminal tagged HIMF was generated by PCR andinserted into pcDNA5/FRT/TO vector using the EcoR V and Xho Irestriction sites. The pcDNA5/FRT/TO vector containing the recombinantHIMF gene was then integrated in a Flp recombinase-dependent manner intothe genome of the Flp-In™ T-REx™ 293 cell line using the Flp-In™ T-REx™kit from Invitrogen (Carlsbad, Calif.). Production of recombinant HIMFwas induced by tetracycline (1 μg/ml) in the 293 cells maintained inDMEM supplemented with 5% FBS, 2 mM of glutamine, 100 U/ml of penicillinand 100 μg/ml of streptomycin, 100 μg/ml of hygromycin B, and 7.5 μg/mlof blasticidin. HIMF was purified by anti-FLAG M2 antibody agarose(Sigma, St. Louis, Mo.) column chromatography from the 293 cell culturemedium with FLAG (0.1 mg/ml) elution. The concentration of the HIMFprotein was estimated semi-quantitatively by silver staining afterSDS-PAGE using BSA as the standard. N-terminal sequencing of therecombinant protein revealed that its N-terminal sequence isDETIEIIVENKVKEL; (SEQ ID NO: 9), suggesting mature HIMF protein has 88amino acids. Therefore, the recombinant protein has 96 amino acids andhas a calculated molecular weight of 10,444 Da. Preliminary experimentsrevealed that the recombinant protein activated Akt in both RPSM and L2cells. To exclude the role of possible contaminants such as insulin, amock isolation control experiment was performed. The medium obtainedfrom the control TREx-293 cell culture was used for the mock isolation.The FLAG elution from the HIMF isolation or the mock isolation wereconcentrated and washed extensively with PBS by the 20 ml centrifugefilter (3000 MWCO). The HIMF isolation stimulated Akt phosphorylation inthe L2 cells. The mock isolation, however, had no effect. The resultsdemonstrated that the effect is HIMF specific and that the recombinantHIMF does not contain insulin, a strong Akt activator. There areconflicting reports about whether or not HIMF exists at higher orderforms (dimer or polymer). Banerjee and Lazar reported that recombinantHIMF (RELMα) could not form a dimer3. However, Holcomb et al. showedthat the native HIMF (FIZZ1) in the lung homogenate formed higher orderforms⁴, and Blagoev et al. reported that recombinant HIMF (RELMα) alsoformed higher order forms5. Our result revealed that HIMF indeed mayexist in a dimeric form.

Western blots. The tissue collection and homogenization were performedas described before¹. HIMF was detected with 1:1000 dilution of theanti-HIMF antibody (raised in rabbit using the peptide CENKVKELLANPANYP(SEQ ID NO: 10) conjugated to keyhole limpet hemocyanin as the antigen)followed by 1:3000 dilution of goat anti-rabbit HRP-labeled antibody(BioRad, Hercules, Calif.). The specificity of the anti-HIMF antibodywas preliminarily characterized with peptide competition. The peptideused as the immunogen blocked only the HIMF band and had no effect onthe nonspecific bands. For phospho-Akt (pAkt) and Akt blots, RPSM werelysed in CelLytic-M (Sigma, St. Louis, Mo.) supplemented with 1 mM PMSFand 1:100 of protease inhibitor cocktail, phosphatase inhibitor cocktail1 and 2 (Sigma, St. Louis, Mo.). 18 μg of protein in equal volume wassubjected to electrophoresis in 4-20% polyacrylamide gel (BioRad,Hercules, Calif.). After transfer to nitrocellulose membrane, themembrane was first probed with anti-pAkt1/2/3(Thr308)(1:500, (Santa CruzBiotechnology, Santa Cruz, Calif.). Then it was stripped and reprobedwith anti-Akt1/2/3 (1:500, (Santa Cruz Biotechnology, Santa Cruz,Calif.).

Immunohistochemistry of HIMF. After the mice were sacrificed, the lungswere fixed in 4% formaldehyde in PBS for 2 hours, and 8 μm frozensections were cut. Following three washes with PBS, the sections wereincubated for 1 hour with the anti-HIMF antibody (1:1000 dilution)followed by a 2-hour incubation with goat anti-rabbit antibodyconjugated with HRP.

Matrigel plug assays: 200 μl of Matrigel mixed with FLAG or HIMF (50 nMfinal) was injected subcutaneously (n=3 for each group) on the back ofmice. Mice were sacrificed after 7 days and the Matrigel plugscollected. Following fixation in 4% paraformaldehyde in PBS (0.1 mM, pH7.4), 10 μm frozen sections were cut and stained either with hemotoxylinand eosin (HE) or von Willebrand factor (vWF). For the detection of vWF,anti-human vWF (1:250 dilution, Dako) and anti-rabbit IgG conjugatedwith FITC (1:500 dilution) were used. The positive cells were countedand expressed as cell numbers per 20× field.

Intact chest mouse experiment. The animals were ventilated with 95%O2/5% CO2 and 2% isoflurane using a custom-designed, constant-flow mouseventilator with tidal volume set to 6.7 ml/kg at 140 breaths/min. Afemoral artery was cannulated for the measurement of systemic arterialpressure, which was measured with a Viggo-Spectramed transducer attachedto a polygraph (Grass Instruments, model 7). Heart rate was determinedfrom the systolic pressure pulses with a tachometer (Grass, model7P44A), and the left jugular vein was cannulated for the administrationof drugs. The animals were strapped in the supine position to afluoroscopic table, and PAP was measured with a specially designedmurine pulmonary arterial catheter attached to a pressure transducer(Schneider/Namic). Mean PAP was derived electronically, and pulmonaryarterial wedge pressure was determined by recently describedprocedures⁶. Cardiac output was measured by the thermodilution techniqueby injection of a known volume (20 μl) of 0.9% NaCl solution at 23° C.into the right atrium and measurement of blood temperature changes inthe root of the aorta with a cardiac output computer (Cardiotherm 500,Columbus Instruments, Columbus, Ohio) equipped with a small-animalinterface. A thermistor microprobe (Columbus Instruments, Fr-1) wasinserted into the right carotid artery and advanced to the aortic arch,where changes in aortic blood temperature were measured.

Rapid amplification of cDNA ends. 5′ and 3′ Rapid amplification of cDNAends (RACE) were performed using the SMART RACE cDNA Amplification Kitfrom Clontech according to the manufacturer's instruction. The messengerRNA isolated from mouse lung was used as the template. Mouse HIMF genespecific primers were designed based on the GenBank sequence (AA712003).5′-TTA GGA CAG TTG GCA GCA GCG GGC AGT G-3′ (SEQ ID NO: 11) and 5′-GATCCA TCA GCA AAG CCA CAA GCA CAC-3′ (nested primer, the underlinednucleotides should be CAG for the positions according to the 3′ RACE;SEQ ID NO: 12) were for the 5′RACE; 5′-TCC CTT CTC ATC TGC ATC TCC CTGCTC C-3′ (SEQ ID NO: 13) and 5′-CTT GCC AAT CCA GCT AAC TAT CCC TCCAC-3′ (nested primer; SEQ ID NO: 14) were for the 3′ RACE. Mus musculuscDNA clone IMAGE:1195776 was obtained from ATCC and was sequenced. ThemRNA sequence obtained by the 5′ and 3′ RACE is identical to the IMAGE1195776.

Cloning of mouse HIMF genomic sequence. 4,159 bp of mouse HIMF genomicsequence was cloned by genome walking using the GenomeWalker kit(Clontech) according to the manufacturer's instruction. The mousegenomic libraries were used as the template. The primers for walking tothe 5′ direction were 5′-TTAGGACAGTTGGCAGCAGCGGGCAGTG-3′ (SEQ ID NO:15), 5′-GATCCACAGGC AAAGCCACAAGCACAC-3′(SEQ ID NO: 16), and5′-GGTCTCATCAGTATTCACTGGGACCATC-3′(SEQ ID NO: 17). And the primers forwalking to the 3′ direction were 5′-CAAGACTATGAACAGA TGGGCCTCCTGC-3′(SEQ ID NO: 18) and 5′-TGACCATGCAGGGATGACTGCTACTGG-3′(SEQ ID NO: 19).The mouse HIMF genomic fragments were cloned into pCR2.1 (Invitrogen)and were sequenced. 4,159 bp of the genomic sequence was assembled andthe exon/intron boundaries were determined. The HIMF gene has fourexons. Exon 1 is 78 bp; exon 2 is 152 bp; exon 3 is 81 bp; and exon 4 is287 bp. The translation starts at exon 2, and stops at exon 4. Thegenomic sequence was deposited into GenBank (AF516926).

Analysis of Nucleic acid motifs. Analyzing the HIMF genomic sequencerevealed multiple hypoxia and inflammation related transcription factorbinding motifs. According to the consensus sequence TKNNGYAAK⁷ (SEQ IDNO: 20), six C/EBP binding motifs were found. Among them four werelocated at the 5′ flanking region, and two were at the 3′ flankingregion. We located five NF-κB motifs (one nucleotide mismatch) accordingto the consensus sequence GGGRHTYYCC⁷ (SEQ ID NO: 21). One was at the 5′flanking region, two were at introns 1 and 2, one was at 3′UTR, and onewas at the 3′ flanking region. Searching the genomic sequence with theconsensus sequence TGASTCAG⁴ (SEQ ID NO: 22), an AP 1 site was found at3′UTR. At the intron 3, a putative IRF site (GAAACCAAAAGT; SEQ ID NO:23) was found matching the consensus sequence GAAASYGAAASY⁷ (SEQ ID NO:24) 11 out of 12. Searching the genomic sequence with the consensussequence TTCNNNNGAA (SEQ ID NO: 25), two GAS elements were found. One islocated at −87 to −78 bp, which has been demonstrated to be the Stat6binding site that mediates the IL4-induced promoter activity in myeloidcell line Bmnot cells⁸. Another is located at intron 2.

Statistical analysis. All results are expressed as mean±SEM, andanalyzed statistically with either Student t-test or ANOVA followed byNewman post hoc analysis as appropriate. Statistical significance wasset as P<0.05.

References for Example 1

-   1. Quinlan, T R, Li, D, Laubach, V E, Shesely, E G, Zhou, N, Johns,    R A. eNOS-deficient mice show reduced pulmonary vascular    proliferation and remodeling to chronic hypoxia. Am J Physiol Lung    Cell Mol Physiol. 2000; 279:L641-L650.-   2. Johnson B A, Lowenstein C J, Schwarz M A, Nakayama D K, Pitt B R,    Davies P. Culture of pulmonary microvascular smooth muscle cells    from intraacinar arteries of the rat: characterization and inducible    production of nitric oxide. Am J Respir Cell Mol Biol. 1994;    10:604-612.-   3. Banerjee R R, Lazar M A. Dimerization of resistin and    resistin-like molecules is determined by a single cysteine. J Biol    Chem. 2001; 276:25970-25973.-   4. Holcomb I N, Kabakoff R C, Chan B, Baker T W, Gurney A, Henzel W,    Nelson C, Lowman H B, Wright B D, Skelton N J, Frantz G D, Tumas D    B, Peale F V, Jr., Shelton D L, Hebert C C. FIZZ1, a novel    cysteine-rich secreted protein associated with pulmonary    inflammation, defines a new gene family. EMBO J. 2000; 19:4046-4055.-   5. Blagoev B, Kratchmarova I, Nielsen M M, Fernandez M M, Voldby J,    Andersen J S, Kristiansen K, Pandey A, Mann M. Inhibition of    adipocyte differentiation by resistin-like molecule 206F α. J Biol    Chem. 2002; 277: 42011-42016.-   6. Champion, H C, Bivalacqua, T J, D'Souza, F M, Ortiz, L A, Jeter,    J R, Toyoda, K, Heistad, D D, Hyman, A L, Kadowitz, P J. Gene    transfer of endothelial nitric oxide synthase to the lung of the    mouse in vivo. Effect on agonist-induced and flow mediated vascular    responses. Circ Res. 1999; 84:1422-1432.-   7. Locker J. RNA polymerase II transcription controls of animals:    DNA binding sites and transcription factors. In: Locker J, editor.    Transcription Factors. Essential Data. Chichester, West Sussex John    Wiley & Sons; 1996: 13-55.-   8. Stutz A M, Pickart L A, Trifilieff A, Baumruker T,    Prieschl-Strassmayr E, Woisetschlager M. The Th2 Cell Cytokines IL-4    and IL-13 Regulate Found in Inflammatory Zone 1/Resistin-Like    Molecule α Gene Expression by a STATE and CCAAT/Enhancer-Binding    Protein-Dependent Mechanism. J Immunol. 2003; 170:1789-1796.

Example 2 Overview

Pulmonary vascular remodeling, characterized by pulmonary microvascularsmooth muscle cell proliferation, is implicated in the development ofhypoxic pulmonary arterial hypertension (PAH). To search for the genesthat may participate in the pulmonary remodeling, a cDNA microarrayanalysis (Incyte Genomics; 9415 genes) was performed using lung samplesfrom mice exposed to 10% O₂ or room air for 4 days. EST AA712003 (FIZZ1)was found to be induced by hypoxia.

We hypothesized that FIZZ1 participates in the process ofhypoxia-induced pulmonary remodeling. FIZZ1 could be induced at or nearthe pulmonary vasculature by hypoxia, and the secreted FIZZ1 might havea proliferative effect on the pulmonary vascular smooth muscle cells. Weproceeded to validate the microarray result and confirmed that FIZZ1 wasmarkedly induced by hypoxia in the pulmonary vasculature as well as inbronchial epithelial cells and type II pneumocytes. We tested theproliferative role of FIZZ1 in cultured rat pulmonary microvascularsmooth muscle cells (RPSM) using a ³H-thymidine incorporation assay withrecombinant FIZZ1. FIZZ1 was shown to stimulate RPSM proliferation, andso we renamed FIZZ1 as hypoxia-induced mitogenic factor (HIMF).

Example 3 HIMF is Induced in Lung by Hypoxia

RT-PCR results confirmed that hypoxia upregulated HIMF mRNA expressionin murine lungs, and the HIMF mRNA induction peaked at 1 day of hypoxiaand lasted for 7 days (FIG. 1A), corresponding with the period ofmaximum vascular smooth muscle cell proliferation during development ofhypoxic PAH.⁷

Western blot shows that exposure of mice to hypoxia for 4 dayssignificantly increased HIMF protein expression only in lung (FIG. 1B).The immunohistochemistry of mouse lung sections showed that, undernormoxic conditions, HIMF protein was not expressed in the pulmonaryvasculature, and its expression in epithelial cells was minimal (FIG.1C). Like the ovalbumin-induced asthma model,¹ exposure of mice tohypoxia for 4 days increased HIMF protein expression in airwayepithelial cells and type II pneumocytes (FIG. 1D). However, unlike theovalbumin-induced asthma model¹, hypoxia markedly increased HIMFexpression in the pulmonary vascular cells. HIMF is a secreted protein¹(FIG. 2A). Therefore, our results suggest that under hypoxia, HIMF maybe secreted from the pulmonary vascular cells as well as the neighboringtype II pneumocytes and act on the pulmonary vascular cells through bothparacrine and autocrine fashions.

We cloned and sequenced the genomic sequence of mouse HIMF gene(AF516926). Multiple inflammation-related transcription factor bindingmotifs such as NF-κB, C/EBP, and GAS were found across the genomicsequence, the 5′ and 3′ flanking regions and introns, suggesting thatthe expression of HIMF may be regulated by those transcription factors.The mechanism by which hypoxia upregulates the expression of HIMF inlung remains unknown and is worth investigating.

Example 4 HIMF Stimulates RPSM Proliferation

Chronic hypoxia exposure will result in pulmonary vascular remodelingthat is characterized by pulmonary vascular smooth muscle cell migrationand proliferation, a major event in the development of hypoxia-inducedPAH. We hypothesized that the hypoxia-induced HIMF expression mightparticipate in the pulmonary remodeling. Using a ³H-thymidineincorporation assay, the effect of recombinant HIMF on the proliferationof RPSM was tested. FIG. 2B shows that HIMF dose-dependently increasedthe proliferation of RPSM at concentrations of 3.3×10⁻⁹ to 3.3×10⁻⁸mol/L, suggesting that HIMF may play a role in the processes ofhypoxia-induced pulmonary vascular remodeling. The exact role of HIMF inthe processes of hypoxia-induced pulmonary vascular remodeling needs tobe evaluated in vivo.

Example 5 PI3K/Akt Signal Transduction Pathway at Least in Part Mediatesthe Proliferative Effect of HIMF

The phosphatidylinositol 3-kinase (PI3K) family of enzymes is activatedby a variety of upstream signals and produces 3′ phosphoinositidelipids, which bind to and activate diverse cellular target proteins thatultimately result in the mediation of cellular activities such asproliferation and survival.⁸ One of the downstream targets of PI3K isthe serine/threonine kinase Aid, which mediates cell growth throughstabilization of cyclin D1 and downregulation of Cdk inhibitors p27 andp21.⁸ The PI3K/Akt pathway has been shown to mediate proliferation andmigration of human pulmonary vascular smooth muscle cells ⁹ and othervascular smooth muscle cells. To test whether the PI3K/Akt pathwaymediates the proliferative effect of HIMF, we first examined whetherHIMF could activate the PI3K/Akt pathway. FIG. 2C shows that HIMFstrongly activated the phosphorylation of Akt. The Akt activation(phosphorylation) peaked at 5 minutes and was sustained through 60minutes. The PI3K inhibitor LY294002 (10 μmol/L) inhibitedHIMF-activated Akt phosphorylation (FIG. 2D). Genistein (20 μmol/L) andNF449 (50 μmol/L), which have been shown to inhibit serum-induced RPSMproliferation in the preliminary experiments, however, did not inhibitthe Akt activation. LY294002 also inhibited HIMF-stimulated RPSMproliferation (FIG. 2E), suggesting that the PI3K/Akt pathway, at leastin part, mediates the proliferative effect of HIMF.

Example 6 HIMF has Angiogenic and Vasoconstrictive Properties

The PI3K/Akt pathway plays a critical role in the regulation of vascularhomeostasis and angiogenesis. HIMF activation of the PI3K/Akt pathwaysuggests that it may have other effects on the vascular cells besidesstimulation of RPSM proliferation. The angiogenic effect of HIMF wasevaluated in an in vivo Matrigel™ plug model. HIMF significantlyincreased the vascular tube formation in the Matrigel™ plug in vivo(FIGS. 3A through 3D), from 4.3±0.84 to 17.7±1.37/×20 field (n=3,P<0.001), suggesting that it is an angiogenic factor. Whether Aktactivation participates in the process of HIMF-induced angiogenesisneeds further investigation.

Intravenous injection of HIMF increased the pulmonary arterial pressure(PAP) and pulmonary vascular resistance (PVR) (FIG. 3E). The molardose-response curves show that HIMF is a more potent pulmonaryvasoconstrictor than endothelin-1, angiotensin II, or serotonin (FIG.3E). Injection of FLAG elution buffer had no effect on PAP, suggestingthat the vasoconstrictive effect is HIMF specific. Thus, HIMF is ahighly potent constrictor of the pulmonary vasculature. The mechanismsunderlying the HIMF-induced constriction and the role of HIMF inpulmonary vascular physiology and pathophysiology require furtherinvestigation.

REFERENCES

-   1. Holcomb I N, Kabakoff R C, Chan B, Baker T W, Gurney A, Henzel W,    Nelson C, Lowman H B, Wright B D, Skelton N J, Frantz G D, Tumas D    B, Peale F V Jr, Shelton D L, Hebert C C. FIZZ1, a novel    cysteine-rich secreted protein associated with pulmonary    inflammation, defines a new gene family. EMBO J. 2000; 19:4046-4055.-   2. Steppan C M, Bailey S T, Bhat S, Brown E J, Banerjee R R, Wright    C M, Patel H R, Ahima R S Lazar M A. The hormone resistin links    obesity to diabetes. Nature. 2001; 409:307-312.-   3. Steppan C M, Brown E J, Wright C M, Bhat S, Banerjee R R, Dai C    Y, Enders G H, Silberg D G, Wen X, Wu G D, Lazar M A. A family of    tissue-specific resistin-like molecules. Proc Natl Acad Sci USA.    2001; 98:502-506.-   4. Raes G, De Baetselier P, Noel W, Beschin A, Brombacher F,    Hassanzadeh G G. Differential expression of FIZZ1 and Ym1 in    alternatively versus classically activated macrophages. J Leukoc    Biol. 2002; 71:597-602.-   5. Rajala M W, Lin Y, Ranalletta M, Yang X M, Qian H, Gingerich R,    Barzilai N, Scherer P E. Cell type-specific expression and    coregulation of murine resistin and resistin-like molecule-α in    adipose tissue. Mol Endocrinol. 2002; 16:1920-1930.-   6. Blagoev B, Kratchmarova I, Nielsen M M, Fernandez M M, Voldby J,    Andersen J S, Kristiansen K, Pandey A, Mann M. Inhibition of    adipocyte differentiation by resistin-like molecule α. J Biol Chem.    2002; 277:42011-42016.-   7. Quinlan T R, Li D, Laubach V E, Shesely E G, Zhou N, Johns R A.    eNOS-deficient mice show reduced pulmonary vascular proliferation    and remodeling to chronic hypoxia. Am J Physiol Lung Cell Mol    Physiol. 2000; 279:L641-L650.-   8. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield M D.    Cellular function of phosphoinositide 3-kinases: implications for    development, homeostasis, and cancer. Annu Rev Cell Dev Biol. 2001;    17:615-675.-   9. Goncharova E A, Ammit A J, Irani C, Carroll R G, Eszterhas A J,    Panettieri R S, Krymskaya V P. PI3K is required for proliferation    and migration of human pulmonary vascular smooth muscle cells. Am J    Physiol Lung Cell Mol Physiol. 2002; 283:L354-L363.

1. A method of treating a patient with hypertrophic cardiomyopathy,comprising: administering to the patient an antibody which specificallybinds to a protein comprising the sequence of SEQ ID NO: 1, in an amountsufficient to reduce the extent of hypertrophic cardiomyopathy.